key: cord-0281757-dfkozriy
authors: Fuentes, Esther; Sivakumar, Niruja; Selvik, Linn-Karina; Arch, Marta; Cardona, Pere Joan; Ioerger, Thomas R.; Dragset, Marte Singsås
title: Drosophila melanogaster is a powerful host model to study mycobacterial virulence
date: 2022-05-12
journal: bioRxiv
DOI: 10.1101/2022.05.12.491628
sha: 11ae702682e4755eb127713cb4192e572b745f72
doc_id: 281757
cord_uid: dfkozriy

Drosophila melanogaster (Drosophila), the common fruit fly, is one of the most extensively studied animal models we have, with a broad, advanced, and organized research community with tools and mutants readily available at low cost. Yet, Drosophila has barely been exploited to understand the underlying mechanisms of mycobacterial infections, including those caused by the top-killer pathogen Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether Drosophila is a suitable host model to study mycobacterial virulence, using Mycobacterium marinum (Mmar) to model mycobacterial pathogens. First, we validated that an established mycobacterial virulence factor, EccB1 of the ESX-1 Type VII secretion system, is required for Mmar growth within the flies. Second, we identified Mmar virulence factors in Drosophila in a high-throughput genome-wide manner using transposon insertion sequencing (TnSeq). Of the 181 identified virulence genes, the vast majority (91%) had orthologs in Mtb, suggesting that the encoded virulence mechanisms may be conserved across Mmar and Mtb. Finally, we validated one of the novel Mmar virulence genes we identified, a putative ATP-binding protein ABC transporter encoded by mmar_1660, as required for full virulence during both Drosophila and human macrophage infection. Together, our results show that Drosophila is a powerful host model to study and identify novel mycobacterial virulence factors relevant to human infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the world's deadliest infectious disease (ongoing SARS-CoV-2 pandemic excluded) with one death every three minutes (1) . Following the World Health Organization (WHO)'s guidelines, drug-sensitive TB is treated with a combination of four antibiotics given for at least six months (1) . Such a long regimen is particularly difficult in countries with socioeconomic issues. The lack of financial 3 resources for sufficient follow-up may lead patients to finish their cures prematurely, nurturing the development of drug resistant Mtb, provoking even longer, more difficult treatment regimens associated with potentially severe side effects. Attractive novel strategies for improved treatments and to reduce the development of Mtb resistance comprise targeting hostpathogen interactions (HPIs), that is, targeting either the pathogen's virulence by disarming it rather than targeting its viability (2) , or targeting the host response factors by enhancing host protection or interfering with host factors enabling infection (called host-directed therapy) (3) .

Hence, we need representative animal models for dissecting HPIs. Drosophila melanogaster (Drosophila) has been proposed to be particularly fit for this purpose (4, 5) .

Drosophila, also known as the common fruit fly, has been fundamental to our understanding of molecular mechanisms in human biology and disease, sharing 60% of its DNA with us (6).

Drosophila's immunity largely depends on the phagocytosis of invading pathogens by plasmatocytes (macrophage-like cells), followed by activation of the Toll or IMD (for IMmune Deficiency) pathways for antimicrobial peptide production (7) . It was the identification of Drosophila's Toll cascade that led to the characterization of human toll like receptors (TLRs) (8) , reshaping the understanding of our own innate immune system. Drosophila do not possess adaptive immunity, creating the opportunity to specifically study the innate immune responses in an isolated yet in vivo setting. Being a work-horse in basic biological research, the (11) (12) (13) . Drosophila has also been useful in assessing antimycobacterial drug activity, infecting the fly with either Mmar or M. abcessus (14, 15 ).

Actually, the use of Drosophila in mycobacterial research was recently summarized and reviewed, emphasizing Drosophila's untapped potential to study mycobacterial HPIs (4).

However, to fully exploit Drosophila for this purpose, we need to understand which mycobacterial virulence genes and mechanisms are at play during Drosophila infection.

Here we show that Drosophila is a powerful model to study and identify mycobacterial virulence genes. By transposon insertion sequencing (TnSeq) we identified, in a genome-wide manner, 181 Mmar virulence genes in Drosophila. Interestingly, over 90% of the genes had orthologs in Mtb, whereas 42% of these again were identified as Mtb virulence genes during previously published mouse model TnSeq screening (16, 17) , suggesting the Mmar-Drosophila infection model may be relevant to better understand Mtb virulence in mammals. Finally, we knocked out one of the identified novel virulence genes, mmar_1660 (a conserved putative ATP-binding protein of an ATP-binding cassette (ABC) transporter), and demonstrated that the mutant was attenuated for growth within Drosophila as well as human macrophages.

The established mycobacterial virulence factor EccB1 is required for Mmar virulence in

We wanted to investigate whether Drosophila is a suitable host model to study mycobacterial virulence and hypothesized that, if so, established mycobacterial virulence factors should be required for Mmar infection in Drosophila. Hence, we infected Drosophila with an Mmar mutant in EccB1. EccB1 (encoded by eccB1) is a core component of the 6 kDa early secretory antigenic target (ESAT6) Type VII secretion system 1 (ESX-1) (18) . ESX-1 secretion enables transport of specific protein substrates involved in Mtb virulence, like CFP-10 and ESAT-6, across the complex mycobacterial cell wall, and is considered a hallmark in mycobacterial virulence (19) . Indeed, a mutation in Mmar eccB1 (transposon insertion mutation; eccB1::tn) Drosophila is a suitable host model to screen for novel mycobacterial virulence genes.

Next, we asked whether Drosophila is suitable to identify novel mycobacterial virulence genes.

We and others have, by TnSeq, previously identified mycobacterial virulence genes in a genome-wide manner using mouse (16, 17, 20, 21) , cattle (22) , or ameba (23, 24) as host infection models. Discovery of virulence genes by TnSeq is based on infecting the host with a bacterial high-density transposon (tn) mutant library, followed by harvesting the library for analysis after infection, comparing its mutant constituents to the input library using massive 6 parallel sequencing (25) . Mutants that are growth-impaired within the host, but not on standard agar medium, are considered virulence genes. Hypothetically, though, a mutant deficient in for instance ESX-1 secretion could be rescued by mutants sufficient in ESX-1 secretion residing within the same plasmatocyte. To investigate whether this could obscure our screen for virulence genes when we pass a high number of mutants through one single fly, we infected Drosophila with 5000 colony forming units (CFU)/fly of either Mmar wt, eccB1::tn mutant or a 1:1 mix of Mmar wt and eccB1::tn mutant. By using an Mmar wt strain carrying a plasmid conferring hygromycin resistance and dsRed expression, wt (dsRed), in combination with the eccB::tn mutant carrying kanamycin resistance encoded within the tn insert, we could by antibiotic selection separate the growth of the mutant from wt growth during co-infection. As seen in Figure 1B , the eccB1 mutant was still growth-attenuated within the flies when coinfected with Mmar wt, while the co-infected flies died similarly to those infected by wt only (Fig. 1A) . The strains grew comparably in 7H9 liquid medium, and the wt (dsRed) strain grew similarly to the wt strain (Fig. 1C) . From this we take that virulence mutants are not rescued by co-infection with other mutants intact in their version of the virulence gene in question, although this may vary depending on the virulence mechanism. Finally, by day seven post infection the wt bacteria had doubled approximately seven times, providing room to differentiate between mutants in virulence and non-virulence genes during TnSeq screening ( Fig. 1A) . Taken together, our results suggest that Drosophila is a suitable host model to screen for novel mycobacterial virulence.

We aimed to identify novel Mmar virulence genes in Drosophila. Hence, we constructed, using ϕMycoMarT7 (26) , and sequenced a high-density transposon insertion library in the E11 strain.

The obtained library contained mutants in 80% of TA sites covering 96.5% of the genes. 7 Moreover, we uncovered 430 essential (E) genes, 130 genes conferring growth defect (GD) when disrupted, 4314 non-essential (NE) genes, and 79 genes conferring growth-advantage (GA) when disrupted (S1 Datasets A). Combining ES and GD categories, the 560 genes that are essential or cause a growth-defect when disrupted in are line with what has been observed in Mtb (27, 28) . In a previous TnSeq study of Mmar (24) , 300 genes were identified as being essential in vitro, of which 82% (247) are also in the ES or GD category in our data (S1 Datasets A).

To specifically identify Mmar virulence genes during Drosophila infection, we passed the generated library through Drosophila (250 flies, 5000 CFU/fly). We subjected the input and output libraries to TnSeq (the latter harvested from the flies seven days post infection) and

found 181 genes that were required for optimal growth within the flies, based on a permutation test ("resampling analysis" (29)) of the difference in mean tn insertion counts per gene in libraries that had undergone fly infection (output) versus libraries grown under in vitro condition (input) (log fold change <0 and adjusted P-value <0.05) (S1 Datasets B). Among the virulence genes identified were those encoding established mycobacterial virulence factors, like phthiocerol dimycoceroserate (PDIM, a cell wall lipid, mmar_1667, _1770-1771), components of the ESX-1 secretion system (10 genes genes between mmar_5399-5459), and the LytR-CpsA-Psr domain-containing protein CpsA (mmar_4966), but also novel genes never before associated with virulence. We also identified genes (15) that conferred Mmar growth advantage within Drosophila when disrupted (log fold change >0 and adjusted P value <0.05), making them the mere opposite of virulence genes (S1 Datasets B). An example of these are the eccA1 (mmar_5443). Our result taken together, we identified 181 mycobacterial virulence factors during Mmar infection of Drosophila.

The relevance of Mmar virulence in Drosophila to Mtb infection.

To investigate the potential relevance the Mmar virulence genes in Drosophila may have to Mtb infection, we compared them to the entire gene pool of Mtb. We found that most of them (90%) had orthologs in Mtb (Fig. 2) . Moreover, when we compared the Mmar virulence genes to those identified previously during Mtb mouse infection (16, 17) , 42 % of them had Mtb orthologs that were also required for virulence in mice. The 88 Mmar virulence genes that did Mmar_1660 is a novel mycobacterial virulence gene.

To validate our screen, we created targeted knock out mutation of one of the novel virulence genes we identified, mmar_1660, ortholog of Mtb rv3041c. This gene encodes a putative conserved ATP-binding protein ABC transporter, MMAR_1660 (30) . When we infected Drosophila with Mmar wt, Mmar Δ1660, and Mmar Δ1660 complemented strains, Δ1660infected flies survived on average one day longer than wt-infected (Fig. 3A) . Complementing the knockout mutant with expression of the intact mmar_1660 gene from a genomic integration site, partially restored the attenuated phenotype of the Δ1660 mutant (Fig. 3A) . All strains grew comparably in vitro in standard liquid medium (Fig. 3B) . These results validate mmar_1660 as a virulence gene during Drosophila infection.

Further, we wanted to investigate whether mmar_1660 contributed to virulence in human macrophages. Therefore, we infected human induced pluripotent stem cells (iPSC)-derived macrophages with the Mmar wt, Mmar Δ1660, and the Mmar Δ1660 complemented strains.

The macrophages survived longer when infected with the Δ1660 mutant compared to wt, as evident from measuring 1) tryphan blue staining of dead macrophages (Fig. 4A) , 2) lactate dehydrogenase (LDH) release from dead macrophages (Fig. 4B) , 3) microscopic visualization of infected macrophages (Fig. S1) , and 4) indirectly by the number of Mmar CFU released into the cell culture medium over the course of infection (Fig. 4C) . Complementary, the intracellular growth of Mmar Δ1660 was impaired compared to wt (Fig. 4D) . We saw a partial rescue of the Δ1660-mediated virulence-impaired phenotype by the complemented strain ( Fig. 4B and S1 ).

In summary, we show that mmar_1660 is required for full virulence in Drosophila as well as in human macrophages.

We hypothesized that Drosophila could be a powerful future host model to study mycobacterial virulence, due to its 1) short generation time, 2) general ease to handle, 3) state-of-the-art molecular tools and fly mutants readily available at low cost, and 4) compliance with the 3Rs for a more humane animal research. Drosophila has been fundamental to our understanding of mechanisms underlying human biology, the response to infection included (7, 8) 

Drosophila has previously shown useful to understand the host's responses towards mycobacterial infections (4), we show here that it is a powerful host model to study mycobacterial virulence from the pathogen's perspective as well, making it particularly apt to unravel mechanisms of HPIs relevant to pathogenic mycobacteria.

By TnSeq of Mmar tn libraries before and after Drosophila infection, we found 181 genes that were required for full Mmar virulence. Of these, many were already established mycobacterial virulence genes, while others were novel to the field (42 and 58% respectively, based on Mtb mouse model TnSeq virulence screening). 91% of the identified Mmar virulence genes had orthologs in Mtb, likely reflecting that virulence mechanisms relevant to innate immunity and growth within the macrophage/plasmatocyte are conserved across the pathogenic mycobacterial Mmar and Mtb, and across arthropod (Drosophila) and vertebrate (mouse) host models.

Among the already established mycobacterial virulence genes we identified during the Mmar-Drosophila infection screening, were genes encoding factors involved in macrophage intracellular survival and escape, affirming that Drosophila is apt to study virulence associated to overcoming innate immunity. For instance, PDIM is thought to contribute to phagosomal escape and macrophage exit (31), the ESX-1 secretion system is thought to secrete immune modulating effectors into the phagosome and cytosol in addition to facilitate phagosomal escape (32), while CpsA contributes to evade macrophage killing by inhibiting the lysosomaltrafficking pathway LC3-associated phagocytosis (33) .

We validated mmar_1660 (the ortholog of Mtb rv3041c) as required for full virulence during Drosophila and human macrophage infection. This gene encodes a putative ABC transporter, predicted to be involved in active transport of possibly iron across the membrane (30) . Iron is an essential nutrient for most organisms, including mycobacteria which rely on various strategies to acquire this precious metal during infection (34) , like siderophore and hemophore production to scavenge ferric iron and heme, respectively (35) . mmar_1660 may therefore be involved in a novel mechanism to obtain iron during infection, or it may encode a hitherto unknown component of already known iron uptake mechanisms. In fact, Mmar orthologs of genes known to be involved in Mtb siderophore uptake (irtA, irtB) and biosynthesis (mbtD, mbtG) (36, 37), were detected as virulence genes in our screen, demonstrating that mycobacterial genes with a function in iron uptake may indeed be discovered using

Among the Mmar genes that mediated growth advantage within Drosophila when disrupted, was eccA1 of the ESX-1 secretion system. While several ESX-1 genes were required for full Mmar virulence, the presence eccA1 seem to rather slows down Mmar proliferation within the flies. EccA1 is thought to regulate mycolic acid lipid synthesis (38) , in addition to facilitate ESX-1-mediated secretion of the key mycobacterial virulence factors ESAT-6 and CFP-10 (39). Actually, Weerdenburg et al. found that, on a cell culture level, EccA1 was required for virulence in mammalian but not in protozoan cells (24) . EccA1 may therefore be involved in fine-tuning ESX-1 secretion, and in certain host species its absence may lead to Mmar growth advantage, perhaps by removing a functional brake for secretion.

Here, for the first time, we show that Drosophila is a powerful model to study and identify Drosophila infection, survival and CFU assay. Infections were performed in 3 to 5-days-old flies. Flies were infected using a Nanoject II (Drummond Scientific Company) set to inject 13.8 nl, and glass needles prepared using a PB-7 needle puller (NARISHIGE) and were not exposed to CO2 anesthesia for more than 15 minutes during the process. Bacterial infection stocks were diluted to 5000 CFU/13. Transposon insertion sequencing, TnSeq. The transposon library was harvested and pooled by scraping agar plates with colonies. Total DNA was purified using Masterpure DNA purification kit (Epicentre) and prepared for TnSeq by PCR amplification of transposongenome junctions and adapter ligation as previously described (43) . The samples were sequenced on an Illumina NextSeq 2000, generating around 12-15 million 150+150 bp pairedend reads per sample.

Bioinformatic analysis of TnSeq datasets. The reads were processed using TPP in TRANSIT (44) , which counts reads mapping to each TA dinucleotide site. Beta-Geometric correction was applied to the datasets to adjust for skewness (45) . Essential genes were identified using a hidden Markov model (HMM), incorporated into TRANSIT (44), as in described in more detail previously for M. avium (20) . Virulence genes (comparative analysis between input and output tn libraries; determining statistical differences in sum of tn insertion counts in genes within library selected in vitro versus after infection) were identified using the "resampling" algorithm incorporated into TRANSIT (44) .

Macrophage infection and CFU assay. Human induced pluripotent stem cell (iPSC) were obtained from European Bank for induced pluripotent Stem Cells (EBiSC, https://ebisc.org/about/bank), distributed by the European Cell Culture Collection of Public Health England (Department of Health, UK) and produced into monocytes as previously described (46) . Monocytes were seeded in 96-well plates and differentiated into macrophages in RPMI 1640 with 10% fetal calf serum (FCS) and 100 ng/mL M-CSF (Prepotech, 300-25).

At day six of differentiation, the cells' medium was changed to RPMI 1640 with 10% FCS and the respective Mmar strains at a multiplicity of infection of 1:2 Mmar CFU per macrophage followed by incubation at 30˚C and 5% CO2. Uninfected cells were included as controls. After two hours incubation, the cells were washed once with phosphate-buffered saline (PBS) to remove extracellular bacteria before adding RPMI 1640 with 10% FCS. At 0, 1 and 2 days post infection supernatant was collected and cells were washed once again with PBS before being lysed in PBS with 0.5% Triton X-100 (Sigma-Aldrich). The supernatant and cell lysate were then spotted in a 10-fold dilution series on 7H10 agar plates containing 1.25 ug/mL Amphotericin B (ThermoFisher Scientific, to avoid possible fungal infections). After one week incubation at 30˚C the plates were taken out to count CFUs from dilutions with optimal countable range.

Trypan Blue Exclusion assay. Trypan blue exclusion assay was performed 0, 1, 2 and 3 days post infection to assess cell viability. First, supernatant from infected macrophages was removed and cells were washed once with PBS followed by Accutase (Sigma-Aldrich) treatment for 1 hour at 37˚C to detach adherent cells. As a final detachment step, the bottoms of the wells were scraped in circular motion with a sterile pipette tip. The cell suspension was then carefully mixed with 0.4% Trypan Blue (GE Healthcare) in a 1:1 ratio and loaded onto cell counting slides (NanoEnTek). The slides were immediacy read with Countess™ 3 Automated Cell Counter (Invitrogen) for viability with optimal range set between 1 x 10 5 and 4 x 10 6 cells/mL according to manufacturer's protocol.

LDH release assay. Lactate dehydrogenase (LDH) release from infected macrophages were determined to assess cytotoxicity 0, 1, 2 and 3 days post infection. Supernatant from infected cells were harvested, pelleted down to remove cellular debris and subjected to colorimetric analysis with LDH Cytotoxicity Assay kit (Invitrogen) according to manufacturer's protocol.

Absorbance values for LDH activity were read at detection wavelength 450 nm and reference wavelength 650 nm using iMark™ Microplate Absorbance Reader (Bio-Rad). Mmar E11 wt, wt (dsRed), and eccB1::tn growth in vitro (7H9 medium) as measured by OD600.

Data represent means ± SEM of three technical replica recorded every 24 hours. The red and the green asterisks represent a statistically significant difference (P value <0.05) between wt and Δ1660-infected macrophages and between Δ1660 and Δ1660c-infected macrophages, respectively, as calculated by Mann-Whitney U test (one-tailed) using GraphPad Prism 9. (C, D) Bacterial burden was assessed by measuring CFU from supernatant (C) and lysed cells (D). Data represents means ± SEM from three technical replicates per condition.

The red asterisk represents a statistically significant difference (P value <0.05) between wt and

For CFU in the supernatant (C), the statistical difference was calculated using the detection limit value for Δ1660-infected macrophages

Δ1660, or Δ1660 complemented (Δ1660c) 0 and 3 days post infection, using EVOS™ FL Auto 2 Imaging System (Invitrogen) with autofocus set at first field each area (each area was defined as one well in a 96-well plate

Essential gene analysis: Mmar E11 in vitro genetic requirement (determined by HMM in TRANSIT (44)). (B) Virulence gene analysis: Mmar E11 genes required for infection in Drosophila (determined by resampling analysis in TRANSIT (44)). Virulence genes = log fold change >0 and adjusted P-value <0

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